It has been possible for some years to determine the concentration of molecular oxygen in a sample by using optical methods based on luminescence quenching. In general, these methods comprise measuring the luminescence intensity and/or the luminescence lifetime of a suitable luminophore, the luminophore being in contact with an oxygen-containing sample and being exposed to illumination.
The basic feature of luminescence quenching is the deactivation of the luminescing excited electronic state of the luminophore taking place on collision with oxygen molecules. As the average number of luminophore molecules in the excited electronic state is reduced by the interaction with the oxygen molecules, the luminescence intensity and the excited state lifetime of the luminophore are reduced. The magnitude of the reduction is connected with the number of oxygen molecules in contact with the luminophore through the Stern-Volmer equation EQU M.degree./M=1+K.sub.sv [O.sub.2 ]
see e.g. IUPAC Commission on Photochemistry, and "Glossary of Terms used in Photochemistry, part III", EPA Newsletter, July 1986. M.degree. and M of the above equation designate the luminescence intensity or the excited state lifetime of the luminophore in the absence and presence of oxygen, respectively. [O.sub.2 ] designates the concentration of molecular oxygen corresponding to the M-value measured. K.sub.sv is the socalled Stern-Volmer constant explained in the above reference. By using this equation and correlating it to samples of known oxygen concentration, it is possible to determine the oxygen concentration of a sample.
A great deal of research has been going on throughout the years to develop and improve the luminescence quenching method of determining oxygen concentrations. The aim of the research has among other things been to find useful luminophore substances, to improve the contact between the luminophore and the oxygen molecules and to develop improved devices suited for specific uses.
Aromatic molecules have been found useful as luminophore substances by several researchers (Stevens in U.S. Pat. No. 3,612,866, Stanley et al in U.S. Pat. No. 3,725,658). Specific examples of useful luminophores are pyrene butyric acid (Longmuir, I. S. and Knopp J. A., "Measurement of tissue oxygen with a fluorescent probe", Journal of applied physiology, 41, 4, USA 1976), porphyrins and derivatives thereof (Kahil in international publication No. WO 87/00023) and inorganic metal complexes (Bacon, J. R. and Demas, J. N. in UK patent application No. 2,132,348). The luminophore may be embedded in a matrix such as a glass matrix as disclosed by Bergman, I., Nature 218, 1968, p. 376, or a polymer matrix as disclosed by Kahil, supra, or Bacon, supra. The method of luminescence quenching has been employed in a variety of systems. For instance, fibre optic probes containing a dye and being implantable in human body tissue are disclosed by Peterson et al. in U.S. Pat. No. 4,476,870.
Further, the optical quenching method is disclosed inter alia in:
Vauthan, W. M. and Weber, G., "Oxygen Quenching and Pyrenebutyric Acid Fluorescence in Water. A Dynamic Probe of the Microenvironment", Biochemistry, Volume 9, No. 3, Feb. 3, 1970, pp. 464-473. PA1 Buckles, R. G. in U.S. Pat. No. 4,399,099, PA1 Murray, R. C., Jr. and Lefkowitz, S. M. in European Patent Publication No. 0 190 829, PA1 Hirschfeld in U.S. Pat. No. 4,542,987, PA1 Lubbers et al in U.S. reissued patent No. Re. 31,879, PA1 Hesse, H. C. in DD patent application No. 106086 and PA1 Murray, R. C., Jr. and Lefkowitz, S. M. in European patent publication No. 0 190 830. PA1 Parker et al in U.S. Pat. No. 4,592,361, PA1 Parker et al in U.S. Pat. No. 4,576,173, PA1 Ogilby, P. R. et al, "The Photosensitized Production of Singlet Molecular Oxygen (.sup.1 .DELTA..sub.g O.sub.2) in a Solid Organic Polymer Glass: A Direct Time-resolved Study", J. Am. Chem. Soc., 109, 1987, pp. 4746-4747. PA1 .phi..sub.X =the quantum yield of the formation of the sensitizing excited state, D*(X), ##EQU1## k.sub.et being the rate constant for energy transfer from sensitizer to ground state molecular oxygen, k.sub.X being the rate constant for decay of the sensitizing excited state ##EQU2## .tau..sub.X being the lifetime of the sensitizing excited state), [O.sub.2 ] being the concentration of molecular oxygen in the matrix containing the sensitizer. PA1 k.sub.et : The rate constant for energy transfer from sensitizer to ground state molecular oxygen. For essentially all potential sensitizers this process is diffusion-controlled. Hence, k.sub.et will be proportional to the sum of the diffusion constants of molecular oxygen and sensitizer in the matrix concerned. PA1 [O.sub.2 ]: For a given ambient partial pressure of molecular oxygen the concentration of molecular oxygen in the matrix (i.e. the matrix containing the sensitizer) will be proportional to the solubility of the molecular oxygen in the matrix concerned. PA1 k.sub.X : The rate constant for decay of the sensitizing excited state (in the absence of oxygen) is largely determined by the choice of sensitizer. Smaller effects of matrix and concentration of sensitizer may occur. PA1 .phi..sub.max being the maximum yield of singlet oxygen luminescence obtainable in the system in question. PA1 modifying the sensitizing excited state lifetimes, i.e. adjusting k.sub.X, PA1 adjusting or modifying k.sub.et [O.sub.2 ] PA1 1) It has fluorescence. PA1 2) The fluorescence is well separated from 1270 nm. PA1 3) The fluorescence has a lifetime which is sufficiently short to make the fluorescence quantum yield independent of oxygen.
The present invention provides a new method of optically determining the concentration of molecular oxygen present in a sample.
When using the method of the invention, the concentration of molecular oxygen in a sample is determined by exciting oxygen molecules of the sample from the electronic ground state to the excited .sup.1 .DELTA..sub.g -state (excited singlet state), measuring a 1270-nm luminescence characteristic of the excited oxygen molecules and correlating the luminescence characteristic measured with the concentration of molecular oxygen in the sample.
In contrast to the basic principle of the known optical methods, i.e. measuring light emitted from another substance than oxygen, the present invention is based on the principles of determining the concentration of oxygen in a sample by measuring the light emission at 1270 nm from oxygen. The method of the present invention has the same advantages as the known optical methods (for example, the oxygen is not consumed in the method of the invention), but is superior to the known methods from a practical measuring point of view. More specifically, the present method is specific to oxygen as the signal obtained at 12170 nm is substantially attributable to oxygen and generally less sensitive to interference from substances other than oxygen. This is in contrast to the known methods based on luminescence quenching, wherein the intensity or the lifetime reductions caused by other substances cannot be distinguished from the oxygen-derived signal changes. Also, the luminescence signal obtained at 1270 nm increases with increasing concentrations of oxygen starting at essentially zero in the absence of oxygen. This makes amplification of the 1270-nm signal obtained in the presence of oxygen by the method of the invention possible. In contrast, the oxygen-induced signal reduction of the known luminescence quenching methods does not permit any signal amplification. Moreover, calibration of the measurement method according to the present invention is more simple than calibration of the known luminescence quenching methods, as the establishment of a standard curve may be based on only one known oxygen concentration in addition to the reference value which is implicitly known (zero in the absence of oxygen).
Examples of publications which discuss the 1270-nm emission from singlet oxygen are: